Streamlining the chemoenzymatic synthesis of asymmetrical glycans of biological importance

项目摘要 几乎所有的细胞表面和分泌的蛋白质都被共价连接的碳水化合物部分修饰，并且 这些所谓的多糖被认为是蛋白质折叠、细胞 信号、受精、胚胎发生、神经元发育、细胞和细胞的增殖 组织成特定的组织。此外，压倒性的数据支持糖基化与病原体的相关性。 识别、炎症、先天免疫反应和自身免疫性疾病的发展 癌症。由于缺乏明确定义的复杂低聚糖标准，糖科学的进展受到阻碍 它们是制造下一代微阵列所需的，用于开发分析 用于阐明糖结合途径的确定分离多聚糖的精确结构的方案 生物合成，以及作为免疫原产生单抗，用于糖蛋白分离和可视化。 在这个应用中，我们建议开发新的合成策略，这些策略可以很容易地提供大型文库 对称和不对称N-糖链。新的方法将利用现成的起点 并将充分标准化，使包括合成服务单位在内的许多实验室能够 采用这些方法。此外，新方法的综合原理可以很容易地应用于 制备其他类别的葡聚糖，如O-连接的葡聚糖和人乳寡糖(HMOS)。 新方法将使用一种对称的双天线多糖，可以很容易地从蛋黄中分离出来。 创新的酶转化将被开发出来去对称化这种多聚糖。此外， 重组N-乙酰氨基葡萄糖转移酶(MGAT‘s)将用于将双触角多聚糖转化为三触角多聚糖. 和四天线结构。在后一种转变中，化学修饰的UDP-GlcNAc供体将是 用来暂时防止手臂被酶修饰。使用最新开发的技术来 表达重组哺乳动物糖基转移酶将是新方法的关键特征。至 验证了该方法学的稳健性，将其应用于糖链衍生库的制备 来自人类上呼吸道上皮细胞。得到的葡聚糖将对下一代产品的开发有价值 产生糖链微阵列，以探测这种细胞类型下的碳水化合物-蛋白质相互作用。这个 化学酶方法学的范围将通过开发能够 容易地提供被硫酸酯修饰的高度复杂的不对称葡聚糖。自动化平台 将开发使用新的捕获和释放技术来进一步提高化学酶合成的速度 战略。注意力将集中在离子交换和镍介导的组氨酸结合事件上，以捕获 标记的低聚糖。一种配置了容积式输液机器人的多通道液体输送机器人 点胶系统和冻干机将被用作自动化工具。后一种方法首先将， 用于制备O-连接的多糖和人乳低聚糖。

Project Summary Almost all cell surface and secreted proteins are modified by covalently-linked carbohydrate moieties, and these so called glycans have been implicated as essential mediators of processes such as protein folding, cell signaling, fertilization, embryogenesis, neuronal development, and the proliferation of cells and their organization into specific tissues. Also, overwhelming data supports the relevance of glycosylation in pathogen recognition, inflammation, innate immune responses, and the development of autoimmune diseases and cancer. Progress in glycoscience is hampered by a lack of well-defined complex oligosaccharide standards which are needed for the fabrication of the next generation of microarrays, for the development of analytical protocols to determine exact structures of isolated glycans, for the elucidation of pathways of glycoconjugate biosynthesis, and as immunogens to produce MABs for glycoprotein isolation and visualization. In this application, we propose to develop novel synthetic strategies that can readily provide large libraries of symmetrical and asymmetrical N-glycans. The new methodologies will make use of readily available starting materials and will be sufficiently standardized that many laboratories, including synthesis service units, can adopt these methods. Furthermore, the synthetic principles of the new approaches can easily be applied to the preparation of other classes of glycans such as O-linked glycans and human milk oligosaccharides (HMOs). The new method will employ a symmetrical biantennary glycan that can easily be isolated from egg yolk. Innovative enzymatic transformations will be developed to desymmetrize this glycan. Furthermore, recombinant N-acetylglucosaminyltransferases (MGAT's) will be used to convert a bi-antennary glycan into tri- and tetra-antennary structures. In the latter transformations, chemically modified UDP-GlcNAc donors will be used to temporarily prevent an arm from enzymatic modification. The use of recently developed technology to express recombinant mammalian glycosyltransferases will be a key feature of the new methodology. To validate the robustness of the methodology, it will be applied to the preparation of a library of glycans derived from human upper airway epithelial cells. The resulting glycans will be valuable for the development of the next generation of glycan microarray to probe carbohydrate–protein interactions in the context of this cell type. The scope of the chemoenzymatic methodology will be further extended by the development of methods that can easily provide highly complex asymmetrical glycans that are modified by sulfate esters. An automation platform will be developed to further increase the speed of chemoenzymatic synthesis using novel capture and release strategies. Attention will focus on ion exchange and nickel-mediated histidine binding events for capture of tagged oligosaccharides. A multi-channel liquid handling robot from Chemspeed equipped with a volumetric dispensing system and lyophilizer will be employed as an automation tool. The latter methodology will, at first, be employed for the preparation of O-linked glycans and human milk oligosaccharides.